The glass slides were air-dried and fixed in 4% paraformaldehyde in PBS. After fixation, the slides were digested with proteinase K for 10 min at 37°C, rinsed in PBS, and fixed again in 4% paraformaldehyde for 5 min. After being washed in PBS for 5 min, the slides were dehydrated in a series of ethanol and air-dried. They were then prehybridized in 50% formamide/double-strength SSC for 2 h at room temperature. Then the slides were hybridized with dig-labeled antisense cRNA probes in hybridization solution. Hybridization was performed in a humidified box for 20 h at 48°C. The slides were washed in double-strength, then single-strength, and then 0.1-strength SSC, each twice for 15 min at 40°C. The hybridized probes were detected using an alkaline phosphatase-coupled anti-dig Fab fragment. The color reaction was developed by incubation with NBT and BCIP in color development buffer. The reactions were terminated by immersing the slides in buffer I.
All experiments measuring PA and PAI-I mRNA and their activities were repeated at least three to five times. Each experiment was designed to obtain enough granulosa cells from about 40-46 ovaries of eCG-primed immature rats for the measurements of both tPA and PAI-I mRNAs as well as their activities at the same time for the comparison. A photographic record of tPA or PAI-I activities on the agarose indicator gel was accomplished by using dark-field illumination. ventolin inhalers
The relative amount of specific mRNA was determined by densitometric scanning of autoradiographic films or by quantitation of counts per minute by Phosphorimager. Each value was normalized against the corresponding relative amount of p-actin in the sample. In each experiment, the data are expressed in terms of fold increase in relation to the control group.
Data were analyzed by ANOVA. Differences among groups were detected by Tukey’s multiple-comparison test. Differences were considered significant when p < 0.05.